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Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis, the causal agent of human whooping cough, and B. bronchiseptica, which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica, which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis, which evolved from a B. bronchiseptica-like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1, and cyoABCD1. To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo. No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis-conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens. Author summary: Cytochrome oxidases, critical components for aerobic respiration, have been shown to be vital for pathogenesis and tissue tropism in several bacterial species. However, the majority of the research has focused on facultative anaerobes and infections of microoxic to anaerobic host environments, like the gut. We sought to understand the role of cytochrome oxidases during respiratory infection by Bordetella bronchiseptica, an obligate aerobe, performing the first analysis of cytochrome oxidases in an extracellular respiratory pathogen that we know of. By comparing B. bronchiseptica to the closely related B. pertussis, a strictly human-specific pathogen and the causative agent of whooping cough, we found three cytochrome oxidases that are important for growth and survival within the mammalian respiratory tract. We also found that a bo3-type cytochrome oxidase, predicted to have a low affinity for oxygen and therefore best suited to ambient air levels of oxygen, was sufficient for both the establishment of infection and persistence in the respiratory tract in mice. Our findings reveal the importance of low affinity cytochrome oxidases in respiratory pathogens, and emphasize the need to study the physiology of diverse classes of pathogens. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-[AP.sup.r]) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-[AP.sup.r] derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naive mice. These 2-[AP.sup.r] derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.

Bordetella pertussis, the causative agent of the acute childhood respiratory disease whooping cough, is a human-adapted variant of Bordetella bronchiseptica, which displays a broad host range and typically causes chronic, asymptomatic infections. These pathogens express a similar but not identical surface-exposed and secreted protein called filamentous hemagglutinin (FHA) that has been proposed to function as both a primary adhesin and an immunomodulator. To test the hypothesis that FHA plays an important role in determining host specificity and/or the propensity to cause acute versus chronic disease, we constructed a B. bronchiseptica strain expressing FHA from B. pertussis (FH[A.sub.Bp]) and compared it with wild-type B. bronchiseptica in several naturalhost infection models. FH[A.sub.Bp] was able to substitute for FHA from B. bronchiseptica (FH[A.sub.Bb]) with regard to its ability to mediate adherence to several epithelial and macrophage-like cell lines in vitro, but it was unable to substitute for FH[A.sub.Bb] in vivo. Specifically, FH[A.sub.Bb], but not FH[A.sub.Bp], allowed B. bronchiseptica to colonize the lower respiratory tracts of rats, to modulate the inflammatory response in the lungs of immunocompetent mice, resulting in decreased lung damage and increased bacterial persistence, to induce a robust anti-Bordetella antibody response in these immunocompetent mice, and to overcome innate immunity and cause a lethal infection in immunodeficient mice. These results indicate a critical role for FHA in B. bronchiseptica-mediated immunomodulation, and they suggest a role for FHA in host specificity. inflammation | adhesin | respiratory infection | filamentous hemagglutinin

Results demonstrate ssrA which encodes stable RNA molecule, tmRNA, is involved in the pathogenesis of Salmonella by serving as an attachment site to the bacterium-specific sequences. Data indicate that ssrA is also required by the phage for its growth.